CN110592047B - Novel method for producing feruloyl esterase by fermenting straws with Verbena pyricularis and application - Google Patents
Novel method for producing feruloyl esterase by fermenting straws with Verbena pyricularis and application Download PDFInfo
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Abstract
The invention relates to the field of biotechnology renewable energy, and particularly relates to a pear bag whip fungus and a method for producing feruloyl esterase by fermenting straws and application of the pear bag whip fungus. The invention discloses a method for producing ferulic acid esterase by anaerobic fermentation of straws of Verbena pyricularis (Piromyces CY 1), wherein the Verbena pyricularis (Piromyces CY 1) is preserved in China general microbiological culture preservation and management center, and the preservation numbers are as follows: CGMCC NO. 18141. The invention discloses a penis et testis Pyri Piromyces CY1 which can survive through in vitro passage by preserving, the fermented straw can produce high-activity ferulic acid esterase, and the fermentation process is simple, has low requirement on equipment, is convenient for popularization, and has important industrial application value and development prospect in the industrial field.
Description
Technical Field
The invention relates to the field of biotechnology renewable energy, in particular to a method for producing feruloyl esterase by anaerobic fermentation of leymus chinensis.
Background
The herba Eupatorii chinensis is east meadow of grassland region of continental EurasiaGrasslandAnd one of the important group-establishing species on drought grassland, which is distributed in a wide range from 36 degrees to 62 degrees in north latitude and 120 degrees to 132 degrees in east longitude, and accounts for more than half of China. The leymus chinensis grows well in cold and dry areas, turns green early in spring and withers yellow late in autumn, can provide more green feeds for a longer time, and the quantity generated along with each year is very large. The leymus chinensis is an energy substance and is not reasonably developed and utilized. The existing anaerobic digestion technology has the problems of low technical efficiency and large popularization difficulty.
Lignocellulose is the main component of the leymus chinensis, and the hydrolysis of the lignocellulose is the rate-limiting step in the whole anaerobic digestion and is also a technical difficulty. The lignocellulose biomass mainly comprises cellulose, hemicellulose and lignin, cellulose molecules are embedded in the lignin by covalent bonds combined by the lignin and the hemicellulose, ether bonds and carbon-carbon bonds in the lignin form macromolecular aromatic compounds with a three-dimensional structure, and the strong chemical bonds inhibit the action of hydrolase. Thus, pretreatment of lignocellulose is required. Common methods for pretreating lignocellulose include mechanical methods, heat treatment methods, and chemical treatments, all of which are effective in promoting anaerobic digestion, but these pretreatment methods are costly and not environmentally friendly. The common microbial treatment has more defects, the single microbial treatment effect is not good, the effect of the composite flora of the artificial component is not ideal, and the strains have antagonistic expressions, so that the pretreatment time is long, the conversion efficiency is low, and no complete scheme is provided for producing the ferulic acid esterase by performing anaerobic fermentation on the leymus chinensis.
Dzo is the first generation of the cross between yak and cattle. Dzo (male) and milk cow (female) have obvious hybridization advantages, and the meat and milk production capacity and working capacity are close to those of yak. Wild blood yak frozen semen is used for hybridizing western siemens cattle in rural areas, the filial generation of the wild blood yak frozen semen is dzo, and the dzo contains 50% of wild yak blood, so that the wild yak has high environment adaptability to Qinghai-Tibet plateau. The rumen of dzos inhabits uniquely, complexly and various, a large number of microbial communities synergistically metabolize wild pasture to efficiently degrade so as to provide survival energy and nutrient substances for yaks, and the rumen of dzos becomes an efficient lignocellulose degradation enzyme system through long-term natural selection and evolution, so that the rumen of dzos has unique advantages and efficient lignocellulose degradation capability.
The method for treating the leymus chinensis by adopting the anaerobic fungi is a newer and more effective means. During the period of doctor, the inventor researches the co-culture of the rumen anaerobic fungi and the methane bacteria of the yak and the anaerobic fungi pure culture by taking corn straws, rice straws and wheat straws as substrates to carry out anaerobic fermentation (Weiyaqin, the diversity of the co-culture of the rumen anaerobic fungi and the methane bacteria of the yak and the fiber degradation characteristics research [ D ].2016 ]), evaluating the straw degrading effect of the anaerobic fungus and methane bacterium co-culture and the anaerobic fungus pure culture by detecting gas production, polysaccharide hydrolase activity, various esterase activities, dry matter degradation rate, phenolic acid release amount, methane and acetic acid yield, wherein the activities of the ferulic acid esterase generated by respectively degrading three straws by the anaerobic fungus pure cultures N.frontalis Yak16, Piromyces Yak18 and O.joyonii Yak7 for efficiently degrading the three straws are all lower than 5mU, and the highest activity of the ferulic acid esterase generated by degrading the wheat straws by the anaerobic fungus pure cultures N.frontalis Yak16 is 4.9 mU. The anaerobic fungus Piromyces CY1 separated from the rumen of dzo in the invention produces ferulic acid esterase by anaerobic fermentation with sheep grass as a substrate, the activity reaches 30.1mU, and an unexpected effect is obtained.
Disclosure of Invention
The strain used in the anaerobic fermentation is a pure culture Piromyces CY1 of Pityrosporum ovale which is separated from the rumen content of dzo cattle grazed in Gansu Tianzhu Wuling farm of Qinghai-Tibet plateau, is preserved in the China general microbiological culture preservation management center, and has the preservation number: CGMCC NO.18141, preservation date of 2019.7.9, preservation unit address: the classification name of the Xilu No.1 Hospital No. 3, Beijing, Chaoyang, is: the strain Pityrosporum ovale Piromyces CY 1.
The invention provides a method for producing ferulic acid esterase by anaerobic fermentation of straws of a pure culture of Pitomyces pear CY1, which specifically comprises the following steps:
(1) preparation of pure culture microbial inoculum of Piromyces CY1
Inoculating 10% v/v inoculum size of a pure culture bacterial liquid of Piromyces CY1 into a liquid minimal medium, adding 1% w/v dry and crushed straw as a substrate, simultaneously adding a compound antibiotic for subculture, and placing at 39 ℃ for anaerobic culture for 72h to obtain the high-activity microbial inoculum.
(2) Feruloyl esterase prepared by fermentation
And (2) absorbing the microbial inoculum prepared in the step (1), inoculating the microbial inoculum into the liquid minimal medium which takes 1% w/v straws as a substrate and is the same as the liquid minimal medium prepared in the step (1) according to the inoculation amount of 10% v/v, simultaneously adding the compound antibiotic, and carrying out anaerobic culture at 39 ℃ for 7 days.
Preferably, the liquid minimal medium formula comprises: yeast extract 1.0g, peptone 1.0g, NaHCO37.0g of resazurin (1.0 g/L) 1m L-cysteine hydrochloride 1.7g, 8000 × g of rumen fluid collected before morning feeding, supernatant 170m L obtained after centrifugation at 4 ℃ for 20min, salt solution I165m L, salt solution II 165m L and distilled water to reach the constant volume of 1000m L.
Preferably, the salt solution I comprises 6g of NaCl, (NH4)2SO43g，KH2PO43g，CaCl2·2H2O 0.4g，MgSO4·2H2O0.6g and distilled water to 1000m L.
Preferably, the salt solution II is packagedBuff 4g K2HPO4And distilled water is added to reach the constant volume of 1000m L.
Preferably, the straws added in the step (1) are wheat straws.
Preferably, the straws added in the step (2) are any one of Chinese wildrye, rice straws, wheat straws and corn straws.
Preferably, the substrate is added in the step (2) and then oxygen is removed, and the mixture is sterilized at high temperature and high pressure.
Preferably, the antibiotic is penicillin, streptomycin sulfate and chloramphenicol, and the compound antibiotic is added in the fermentation process, so that the co-culture system can be prevented from being polluted by bacteria and methane bacteria, and the anaerobic fermentation efficiency is improved.
Preferably, the final concentration of penicillin and streptomycin sulfate in the composite antibiotic in the anaerobic culture medium is 1600IU/m L and 2000IU/m L respectively, and the final concentration of chloramphenicol in the culture medium is 50 mu g/m L.
①, the activity of ferulic acid esterase produced by fermenting straws by using the Verbena pyricularis Piromyces CY1 disclosed by the invention is obviously improved, particularly, the sheep grass is used as a substrate for anaerobic fermentation, the activity of the produced ferulic acid esterase is 30.1mU, ②. the Verbena pyricularis can be preserved in vitro for survival and passage, the fermented sheep grass can produce high-activity ferulic acid esterase, the fermentation process is simple, the requirement on equipment is low, the popularization is convenient, and the method has important industrial application value and development prospect in the industrial field. ③. the high-activity ferulic acid esterase can be produced by anaerobically fermenting the sheep grass by using the Verbena pyricularis rumex ruminants.
Detailed Description
The technical solutions claimed in the present invention will be described below with reference to specific examples, but the scope of the claimed invention is not limited to the following examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The anaerobic medium used in the following examples is as follows:
the formula of the liquid minimal medium is as follows: yeast extract 1.0g, peptone 1.0g, NaHCO37.0g of resazurin 1m L-cysteine hydrochloride 1.7g and 1.0 g/L g of 1.7g, 8000 × g of rumen fluid collected before morning feeding, supernatant 170m L obtained after centrifugation at 4 ℃ for 20min, salt solution I165m L, salt solution II 165m L and distilled water to reach the constant volume of 1000m L.
Salt solution I contains 6g of NaCl, (NH)4)2SO4 3g，KH2PO43g，CaCl2·2H2O 0.4g，MgSO4·2H2O0.6g, distilled water to 1000m L.
Salt solution II comprises 4g K2HPO4And distilled water is added to reach the constant volume of 1000m L.
Separating and purifying culture medium, adding 1.0 g/L glucose into liquid minimal medium without straw, then removing oxygen and sterilizing.
Agar roller tube medium 1.0 g/L glucose and 20 g/L agar powder were added to the liquid minimal medium, then deoxygenated and sterilized.
Straw culture medium: adding 1% w/v of pulverized and air-dried herba Leymi, wheat straw, corn straw and rice straw into a liquid minimal medium respectively, without adding glucose, removing oxygen and sterilizing.
Subculture medium: adding 1% w/v of crushed air-dried wheat straw to a liquid minimal medium, then deoxygenating and sterilizing.
The oxygen removing method comprises the following steps: subpackaging the liquid minimal medium into Henschel anaerobic tube or anaerobic bottle, wherein the anaerobic tube or anaerobic bottle is connected with a vacuum pump and high-purity CO via a needle2The air extractor(s) removes oxygen from the culture medium. Firstly, the color of the culture medium is changed when the gas in the vacuum pump extraction pipe reaches the negative pressure, and then high-purity CO is filled in2. And 3 times of air pumping and inflating for each tube, wherein the 1 st time is about 15min, the rest 2 times are 5min each time, the anaerobic tube is inflated for the last 1 time, then the air is deflated again by using a sterile strain needle to balance the internal and external pressures of the anaerobic tube, and then the anaerobic tube is sterilized by moist heat at the high temperature of 121 ℃ for 20min for later use.
Example one, preparation of a Pityrosporum ovale Piromyces CY1 microbial inoculum:
sucking 1m L Verbena pyricularis Piromyces CY1 culture, inoculating into 9m L liquid minimal medium with the volume of 20m L of Henry anaerobic tube and taking air-dried and crushed 0.1g of wheat straw as a substrate, simultaneously adding compound antibiotics, so that the final concentrations of penicillin and streptomycin sulfate in the liquid minimal medium are 1600IU/m L and 2000IU/m L respectively, and the final concentration of chloramphenicol in the culture medium is 50 mu g/m L, carrying out anaerobic culture at 39 ℃ for 72h, and reaching the growth peak, wherein the fermentation liquid is a high-activity microbial inoculum.
EXAMPLE two Feruloyl esterase production by anaerobic fermentation of Leymus verrucosa Piromyces CY1
1. Method for producing feruloyl esterase by anaerobic fermentation of leymus chinensis
The preparation method comprises the steps of filling 90m L of liquid basic culture medium in an anaerobic fermentation bottle with the volume of 100m L, taking 1.0g of crushed and air-dried Chinese wildrye, rice straw, wheat straw and corn straw as substrates, deoxidizing, sterilizing, sucking 10m L of sorangium pyricularis Piromyces CY1 subjected to subculture for 72h by using an aseptic injector, respectively inoculating the sorangium pyricularis Piromyces CY1 into the anaerobic culture medium added with the Chinese wildrye, the rice straw, the wheat straw and the corn straw as substrates, simultaneously adding compound antibiotic, enabling the final concentration of the solution of the anaerobic culture medium to be penicillin 1600IU/m L and streptomycin sulfate 2000IU/m L, enabling the final concentration of the chloramphenicol in the culture medium to be 50 mu g/m L, carrying out anaerobic culture at 39 ℃ for 7 days, totally setting 3 parallel experiments, and measuring the activity of the ferulic acid esterase of the.
2. Method for measuring activity of ferulic acid esterase
The crude enzyme solution was diluted and preheated at 39 ℃ for 15min, a 100mM3- (N-morpholine) propanesulfonic acid (MOPS) solution (pH 6.8) containing 100. mu.M of ferulic acid methyl ester (Sigma Chemicals) as a substrate was preheated at 39 ℃ for 15min, 200. mu. L substrate was added to 100. mu. L crude enzyme solution and reacted at 39 ℃ for 30min, absorbance values at 340nm were measured with a microplate reader (680XR, Bio-rad, USA) for 0min and 30min of reaction, and ferulic acid esterase activity was calculated from the standard curves of ferulic acid and ferulic acid methyl ester.
1 enzyme activity unit U refers to the enzyme quantity required by 1m L enzyme solution to release 1mu mol ferulic acid from a standard substrate ferulic acid methyl ester within 1min under the enzymatic reaction condition.
100mM MOPS buffer (pH 6.8) 5.2323g MOPS was weighed out and dissolved in 150m L deionized water, adjusted to pH 6.8 with NaOH, and made up to 250m L with deionized water.
Experimental results show that the high-activity ferulic acid esterase generated by the Piromyces piricola CY1 when the Piromyces piricola degrades leymus chinensis is obviously more than 10 times higher than the activity of the ferulic acid esterase generated by the Piromyces piricola degrading various straws and other coarse feeds reported in the prior literature and is also higher than the activity of the ferulic acid esterase generated by the Piromyces piricola degrading wheat straws, corn straws and rice straws. The specific results are as follows:
TABLE 7 days of culture period of the Verbena pyricularis Piromyces CY1 Feruloyl esterase activity produced by degrading Chinese wildrye, wheat straw, corn straw and rice straw
The activity of the feruloyl esterase generated by degrading the leymus chinensis through the culture of the Verbena pyrifera Piromyces CY1 reaches the maximum value of 30.1mU within 7 days.
Through the above examples, it can be seen that Piromyces CY1 of Piromyces ruminis of dzos degrades leys aegilops and simultaneously generates high-activity ferulic acid esterase, and the method has important industrial application value and development prospect in the industrial field.
Claims (7)
1. A kind of pear sac fungus: (A)Piromycessp.) CY1 method for producing ferulic acid esterase by anaerobic fermentation of straws, which is characterized by comprising the following steps:
(1) pear scrophularia (B) ((B))Piromycessp.) preparation of CY1 pure culture microbial inoculum
Mixing the pear sac fungus (B)Piromycessp.) inoculating the CY1 pure culture bacterial liquid into a liquid minimal medium at an inoculation amount of 10% v/v, adding 1% w/v dry and crushed wheat straw as a substrate, simultaneously adding a compound antibiotic for subculture, and performing anaerobic culture to obtain a high-activity microbial inoculum;
(2) production of feruloyl esterase by anaerobic fermentation of straws
Absorbing the microbial inoculum prepared in the step (1), inoculating the microbial inoculum into a liquid minimal medium taking 1% w/v straws as a substrate according to the inoculation amount of 10% v/v, and adding compound antibiotics for anaerobic culture;
the described pear sac fungus: (A)Piromycessp.) CY1 is preserved in China general microbiological culture collection center with the preservation number: CGMCC No. 18141.
2. The method of claim 1, wherein the liquid minimal medium formulation is: yeast extract 1.0g, peptone 1.0g, NaHCO37.0g of resazurin 1m L-cysteine salt 1.7g and 1.0 g/L g of resazurin 1m L-cysteine salt, 8000 × g of rumen fluid collected before morning feeding, supernatant 170m L obtained after centrifugation at 4 ℃ for 20min, salt solution I165m L, salt solution II 165m L and distilled water with constant volume of 1000m L.
3. The method of claim 2, wherein said salt solution I comprises 6g NaCl, (NH4)2SO43 g，KH2PO43 g，CaCl2·2H2O 0.4 g， MgSO4·2H20.6g of O, distilled water with constant volume of 1000m L, wherein the salt solution II comprises 4g K2HPO4And distilled water is added to reach the constant volume of 1000m L.
4. The method of claim 1, wherein the antibiotic cocktail is penicillin sodium, streptomycin sulfate, and chloramphenicol at a final concentration of 1600IU/m L and 2000IU/m L in anaerobic culture medium, respectively, and 50 μ g/m L in culture medium.
5. The method of claim 1, wherein the substrate added in the step (2) is any one of wheat straw, corn straw and rice straw.
6. The method of claim 1, wherein the substrate added in step (2) is leymus chinensis.
7. The method of claim 1, wherein in step (2) the straw substrate is added, deoxygenated, and autoclaved.
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